Optical Rotatory Dispersion of L-Amino Acids in Acid Solution

The optical activity of the disulfide bond in L-cystine and some derivatives of L-cystine. David L. Coleman , Elkan R. Blout. Journal of the American ...
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Vol. 3, No.10, October, 1964

OPTICAL ROTATORY DISPERSION

OF AMINO ACIDS

1519

Optical Rotatory Dispersion of L-Amino Acids in Acid Solution* EISAKUI I Z U UAND ~ JENTsI YANG

From the Cardiovascular Research Institute and the Department of Biochemistry, University of California, Sara Francisco Received June 9 , 1964 Optical rotatory dispersion of twenty amino acids in aqueous solution (PH 1)was measured between 190 and 600 mp at 27O. All exhibited a positive Cotton effect near 210 mp, which is perhaps attributable to the carboxyl chromophore attached to the asymmetrical a-carbon. Tyrosine and tryptophan (but not phenylalanine) showed an additional Cotton effect near 275 mp in the absorption band of the aromatic group. Cystine had an unusually high levorotation, an order of magnitude higher than the other amino acids studied. The molar rotations, [MI, above 270 mp can be fitted with a two-term Drude equation: [MI = kl/(Xz - XI2) kz/(Xz M),one accounting for the 210-mp Cotton effect and the other for the remaining partial rotations. The equation was solved graphically by plotting [ M ] ( X ~ - X1*) against l / ( X z - Xz2) for X1 = 210 mp and trial values of Xzuntil a straight line was obtained. In all cases (except tyrosine, tryptophan, and cystine) kl was positive and kz was negative, although the total rotations in the visible region differed widely in both sign and magnitude.

+

Optical rotatory dispersion (ORD) is now extensively used in the conformational studies of proteins and polypeptides (for a recent review, see Urnes and Doty, 1961). It provides a t least a semiquantitative measure of the helicity in the protein molecules, although we are still ignorant of the existence of structural elements other than the a-helix in the molecules and about the complications that could arise because of such secondary and other tertiary structures. To understand better the conformational rotations of the protein molecules, we must also distinguish them from the intrinsic rotations of the amino acid residues, the configurational rotations. In particular, we should like to know how the rotatory properties of individual amino acid residues vary with the nature of the side groups, and if any generalizations about the con@gurational rotations can be made. We studied the ORD of the constituent blocks of the complex protein molecules, the simple amino acids. Although there is no direct one-to-one correspondence between the rotations of a simple amino acid and its residue in a polypeptide chain, any information would help us interpret the ORD of the proteins. The ORD of L-amino acids was studied by many laboratories (Patterson and Brode, 1943; Brand et al., 1954; Otey et d.,1955; Billardon, 1960; Schellman, 1960; Strem et al., 1961; Sasisekharan, 1962; Katzin and Gulyas, 1964. Patterson and Brode (1943) and Otey et al. (1955) reported that the ORD of most of the amino acids obeyed a one-term Drude equation, but Patterson and Brode suggested using the two-term Drude treatment later used by Brand et al. (1954). (Sasisekharan, however, used the squared-type formula [see Discussion] of Chandrasekhar [1952] to fit the data of amino acids.) Levene and Rothen (1936) suggested that the partial rotation of the carboxyl group in an amino acid is probably positive since most Lamino acids show a more positive rotation in acidic than in alkaline solution. We believe the ORD data in the visible region of L-amino acids can be better represented by a two-term Drude equation; one term corresponds to the Cotton effect due probably to the 4 O O H chromophore attached to the a-carbon, and

*This work was supported by grants from the U.S. Public Health Service (GM-K3-3441, GM-10880, and HE06285). 1 On leave from the Ministry of Agriculture and Forestry, under the auspices of the Office of the Prime Minister, Tokyo, Japan. 1 Abbreviation used in this work: ORD, optical rotatory dispersion.

the other represents t.he rest of the partial rotations of the amino acids. Schellman and his co-workers (Schellman, 1960; Strem et al., 1961) classified the amino acids into six groups according to the nature of the side groups: (1) aliphatically substituted side groups, (2) those with chromophores (having an absorption band above 150 mp) beyond the p-carbon, (3) those with p-substituted chromophores, (4) those with more than one asymmetrical center, (5) proline and hydroxyproline, and (6) cystine. All the early studies were confined to the visible region and the more recent ones to a wavelength range above 230-270 m p , and therefore failed to detect experimentally the Cotton effect due to the carboxyl group. Our measurements were extended to about 190 m p . Since the ORD of amino acids is highly dependent on the p H of the medium, but reaches a plateau in strong acids and bases, we restricted our measurements to aqueous solutions having a p H of about 1, where the samples are stable, and, further, where the p H is far away from the p K of the carboxyl groups. EXPERIMENTAL PROCEDURES

Materials.-All amino acids were purchased from the California Corp. for Biochemical Research and used without further purification. The samples were dissolved in 0.1 M HC1 and adjusted to p H 1, and their concentrations were checked by dry-weight method. To extend ORD measurements beyond 200 mp, we used HaSO, instead of HC1 for the p H adjustment. Spectropolarimetry.-The ORD was measured with a Cary Model 60 recording spectropolarimeter a t 27" under constant nitrogen flush. In the visible region the concentrations of the samples used were about 1970, whereas in regions of the absorption bands they were so adjusted that their absorbance was always less than 2 (Umes and Doty, 1961). The instrument calibration has been described elsewhere (Samejima and Yang, 1964). The positioning of the cell on the holder was critical and it could shift the baseline by as much as 0.01 Because of the small magnitude of the measurements for very dilute solutions used, the precision of both the solution and the blank posed a serious problem. This difficulty, however, was minimized by using the thermostable cell assembly which fixed the cell to the holder, rather than the V-shaped holder supplied by the manufacturer. The assembly was not dismantled during the change of solution or solvent. To check further any possible shift of the baseline that may have

".

1520

Biochemistry

EISAKU IIZUKA AND JEN TSI YANG

TABLE I PARAMETERS OF THE DRUDEEQUATIONS FOR L-AM~NO ACIDS(pH

Xz

XI

XC

x

Substance

(mp)

kl X I O d 6

(mp)

Alanine Valine Leucine Isoleucine Serine Threonine Phenylalanine Tyrosine Tryptophan Cystine Cysteine Methionine Proline Aspartic acid Asparagine Glutamic acid Glutamine Histidine Arginine Lysine

210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210 210

+32.6 +23.7 +32.0 $25.7 +23.2 +31.0 +254 -49.4 -31.7 - 170 +67.9 +21.2 $12.7 +12.6 +11.1 1-22.9 +28.1 +51.5 +27.3 +21.7

200 160 180 170 180 185 205 240 260 250 200 170 170 180

-29.3 -16.6 -27.5 -12.6 -19.8 -38.0 - 261 $41.2 +31.7 +10.3 -66.0 -12.3 -35.3 -3.7

180 185 190 185 170

-9.9 -15.7 -47.1 -15.1 -11.3

0

1) ABOVE 270 Mpa

k,

10-6

[M ] N O

(mp)

k X 10-6

256 262 293 235 279

$3.50 +7.34 +3.93 -13.3 +3.66 Anomalous Anomalous Anomalous Nonlinear - 160 +2.25 +9.00 -22.7 $8.82 +11.1 +13.1 1-12.4 $4.50 f12.3 +10.4

+36.9 $80.9 +51.8 +126 +44.3 -34.8 -20.1 -24.0 +68.6 - 1370 +35.2 $89.1 - 159 +79.5 $95.9 121 118 +63.5 116 +lo1

207 314 243 135 223 210 227 234 310 232 236

+ + +

Wavelength Range (600 mp to-) 360 420 400 380 400

290 420 360 380 380 260 360 380 440 380 380

Based on the two-term and one-term Drude equations (4) and (2). I

+4000

-

I

I

. . .. ... : '+-HIS .

i

,

-4000

-

!

ILEL?'

I

I

200

300

I 400

1 500

i J

I

..*I

600 I

I

WAVELENGTH. m p

FIG. 1.-Optical rotatory dispersion of L-amino acids in the visible and near-ultraviolet regions. Abbreviations: ALA, alanine; ILEU, isoleucine; PRO, proline; GLU, glutamic acid; and HIS, histidine.

arisen during the runs, the rotations of a more concentrated solution of any sample (usually ten times that used in the ultraviolet region) was measured in a 10-cm cell for the visible region that overlapped portions of the ultraviolet. The results from both 1-cm and 10-cm cells were compared, and the blank for the 1-cm cell was slightly adjusted, if necessary, so that the overlapping portions gave identical specific rotations, assuming there was no concentration dependence of the measurements. With these precautions the data were

reproducible. (For measurements below 200 mp, we used a specially designed 1-mm cell.) We also found that the warm-up of the instrument took about 1.5 hours after the lamp was on. However, if the coolant and modulator switches were turned on 1 hour or more prior to the ignition of the lamp, the warming-up period could be reduced to about 0.5 hour. All measurements are expressed in terms of molar rotation, [MI,which is equal to [a]X M/100 ( M being the molecular weight of the sample).

3, No. 10,October, 1964

OPTICAL ROTATORY DISPERSION

OF AMINO ACIDS

1521

+ 40

t

: p:

-2000-

- 400

1

t-

200

1-4900

300

400

500

6Cd

WAVELENGTH. m p

- 4000 -

c

. ..

c Y ( ssI

I

I

I

1

200

250

300

350

WAVELENGTH. m p

FIG.3.-Optical

rotatory dispersion of L-amino acids in the visible and near-ultraviolet regions. Abbreviations: THR, threonine; TYR, tyrosine; CY (SH), cysteine; CY(=), cystine; andMET, methionine. Note that the [MI of cystine i s ten times that indicated on the left-hand ordinate.

RESULTS The ORD of half the twenty L-amino acids are shown in Figures 14over a wavelength range of 190400 mp. Table I summarizes the numerical analyses of the experimental results. The strong positive Cotton effect that exists near 210 mp in every case studied (Figs. 2 and 4) can probably be attributed to the presence of the carboxyl chromophore attached to the asymmetrical a-carbon. In most cases the peak of this Cotton effect appeared around 225 mp and the trough near 193 mp (except cystine, which has a trough a t 206 mp). In several cases the crossovers (zero rotations) were near 212 mp. The magnitude and even the sign of the peak and trough rotations differ widely because of the significant differences in the partial rotations of the side groups. The profle of ORD of all amino acids except tyrosine and tryptophan above 270 mp was plain and featureless, even though they can be either dextro- or levorotatory in the visible region. Tyrosine and tryptophan have an additional positive Cotton effect a t 275 mp, which is attributed to the absorption band of the aromatic group. (Schellman, 1960, and Strem etal., 1961, reported that tryptophan in acid solution was levorotatory in the visible region, whereas we found it dextrorotatory. The reason for this discrepancy is not clear to us.) In contrast, the phenyl chromophore of the phenylalanine seems optically inactive. Between 245 and 275 mp the dispersion curve of phenylalanine was not very smooth but was somewhat wavelike. The maximum deviation from the smooth curve was less than 10%. Whether these waves were actually weak Cotton effects or merely artifacts owing to diminution of light intensity in the absorption band was difficult to ascertain. Whatever Cotton effects of phenylalanine might exist near 270 mp, they are too weak to be defined by ORD. (We are unable to confirm Billardon's [ 1960] data on phenylalanine which showed rather strong Cotton effects near 270 mp.) Among the amino acids studied, Lcystine is distinguished by its unusually large levorotation, an order of magnitude larger than that of the

FIG. 4.-Ultraviolet rotatory dispersion of L-amino acids. Abbreviations and [MI of cystine same as in Fig. 3.

other amino acids (see also Turner et al., 1958; Wiirz and Haurowitz, 1961). In contrast, L-cysteine has an ordinary amino acid dispersion. The extraordinary levorotation of L-cystine is thought to be attributable to the intrinsic asymmetry of the disulfide linkage. It has a dihedral angle of 90" and the rotation about the bond is strongly hindered (Calvin, 1954). Indeed, Schellman (1960) suggested that the minimum rotation near 250 m p represented the trough of a Cotton effect. Another plausible explanation is that the apparent trough is the beginning of the positive 210-mp Cotton effect that bends the levorotation toward the dextrorotations. Dr. J. Brahms (private communication) has just informed us that L-cystine at p H 1has a very weak and almost unmeasurable negative circular dichroic band near 250 mp, and a strong positive one a t 222-224 mp (because of instrument limitation his measurements are not yet extended below 220 mp). Note that the Cotton effect of cystine (Fig. 4) has an inflection point near 218 mp, which is close to Brahms' finding. The shift of the trough from 193 mp (for the carboxyl chromophore) to 206 mp might reflect the result of more than one Cotton effect. On the other hand, the extremely weak circular dichroic band at 250 mp accounts for our failure to detect a distinctive Cotton effect in the absorption band of the disulfide linkage. DISCUSSION The wavelength, A, dependence of the molar rotation, [MI, of an optically active molecule, in regions far from the optically active absorption bands, can be described by the general Drude equation [MI =

Zk,/(X* -

Xi2)

(1)

where ki is a parameter proportional to the rotational strength of the ith absorption band with a maximum a t A,. Formally, it is always possible to expand the terms in equation (1) in inverse powers of ( A z - A L 2 ) and reduce them to a one-term Drude equation, [MI =

k/(X* -

kc*)

(2)

1522

Biochemistry

EISAKU IIZUEA AND JEN TSI YANG

TABLEI1 COMPARISON OF THE EXPERIMENTAL AND CALCULATED MOLAR ROTATIONS FOR THREONINE AND TYROSINE (pH ~

Wavelength (mp) 600 540 480 420 360 320 300 270 260 =

~~~~~~

Threonine [MIo a k *

A[Ml

-18.5 -22.2 -27.3 -32.9 -36.1

-18.4 -22.6 -27.3 -32.8 -35.4

-0.1 +0.4 0.0 -0.1 -0.7

-5.5 +94.3 186

-5.9 $93.6 181

$0.4 +0.7 +5.1

+

+

Estimated maximum errors were ~tO.5-0.6'. I

I

-20.4 -23.4 -26.9 -26.7 -6.5 +73.0 237

[na](X2

I

O

Y T

-2

R1 I

-

+

-

X1') = kl

- XL')

=

(KI

+ k2(Xz - h , z ) / ( X 2

+ kz) +

nc

-4

L

z

-6

v

I

I

I

L

I

2

3

4

,0-5/b2

FIG.5.-Graphic

-A:)

solution of the two-term Drude equation for threonine.

provided that the constants k and X, satisfy the conditions Xc2

= ZkiXi'/Zki

(12 = Z k i )

(3)

(Moffitt and Yang, 1956). When equation (2) is not obeyed, the dispersion data can usually be fitted with two Drude terms. Indeed, such a complex dispersion for amino acids has been suggested by Patterson and Brode (1943) and Brand et al. (1954), even when the ORD of amino acids, with a few exceptions, appeared to be simple, i.e., to obey equation (2), over a certain range of wavelength. Results of our studies of twenty amino acids have shown a single positive Cotton effect near 210 mp, the equation describing which is reduced to one Drude term a t wavelengths distant from the absorption band (Moscowitz, 1960). The partial rotations of the other terms in equation (1) can then be approximated by another Drude term. For a two-term Drude equation we will write [MI = k l / ( X z - Xiz)

which can be rearranged as

+ kz/(X'

-

X2')

(4)

h2(Xz2

-

- X2*)

(5a)

(5b) If XI is predetermined, we can plot [M](X2 - Xlz) against (Az - X 1 2 ) / ( X 2 - XZ2) or l / ( X z - XZ2) for trial values of

+2

4

-0.2 +0.3 -0.2 -0.1 -0.8 +0.6 42

+

or

+4

9

A [MI

-20.2 -23.7 -26.7 -26.6 -5.7 +72.4 195

+

[Ml(X2

9

[M I E ~ , I O ~

[M ]expa

* Based on the two-term Drude equation (4).

+6

Y) I

1)

Tyrosine

[Mlexpn

I

'v

~

Xi2)/(Xz

X2 until a straight line is obtained (Fig. 5). Such graphic treatment is reminiscent of the plot employed in the Moffitt equation for polypeptides and proteins (Moffitt and Yang, 1956). The constants K1 and K z are then calculated from the slope and the intercept of the straight line. For amino acids we chose X1 = 210 mp, since the crossovers (Figs. 2 and 4) are located near this wavelength, and even the samples having no crossovers appear to have the same inflection point near 210 mp (except tyrosine, tryptophan, and cystine). The selection of Xl is of course somewhat arbitrary in the absence of circular dichroism measurements. It could slightly shift from one amino acid to another. The use of a common value is simply a matter of convenience. Professor S. Beychok, however, did manage to study an L-leucine solution in 0.1 M HC1 and found a positive and large dichroic band having a peak a t 209210 mp. In another case (isoleucine) we tried to vary X1 and found that either 205 or 215 mp gave a straight line over a shorter range of wavelengths than 210 mp does. Once X1 is fixed, X2 can vary & 5 mp if it is close to X1, but the uncertainty increases to 5 1 0 mp or more for Xz below 170 mp. (Sasisekharan [1962] fitted his data on amino acids with Chandrasekhar's [1952] squared-type formula: [ a ] = KX2/(X2

-

(64

Xo2)2

which is equal to [ a ]=

K/(X2

-

Xo2)

+ KXo'/(X'

-

Xo2)2

(6b)

This is essentially a Moffitt-type equation [Moffitt and Yang, 19561. It is not difficult to show that the two-term Drude equation [4] can be rearranged into equation [6b] through power-series expansion.) The analyses of a l l twenty amino acids using equation (5b) are summarized in Table I. For comparison we also include the constants determined by a one-term Drude equation (2). To check the reliability of such analyses we list in Table I1 the experimental and calculated (from eq. 4) values of two amino acids; threonine gave a very good straight line whereas tyrosine can better be fitted with a three-term instead of a two-term equation (vide infra). In the former case the agreement between the experimental and calculated values is good down to 270 mp, whereas in the latter case it is above 300 mp because of its close approximation of the 275-mp Cotton effect. Several features emerge from

Vol. 3, No. 10,October, 1964

(~PTICAL

Table I. First, with the exception of tyrosine, tryptophan, and cystine, kl is always positive as i t should be for a positive Cotton effect. In most cases (except histidine, phenylalanine, and cysteine) the magnitude of kl varies between 10 and 30 X lo6,which corresponds to a rotational strength of about 2.0-6.0 x 10-40. Second, kz, which principally represents the partial rotations of the side group, is always negative (again with the exception of tyrosine, tryptophan, and cystine) in spite of the fact that the total rotations of the visible region can be either dextro- or levorotatory. Although Schellman (1960) has classified the amino acids into six groups, we found that the twenty amino acids studied can be represented by a two-term Drude equation, one term always positive and the other negative. Tyrosine, tryptophan, and cystine are the exceptions in the sense that the signs of k 1 and k z are reversed as compared with the other seventeen compounds. Two of the three exceptions (tyrosine and tryptophan) are owing to the presence of more than one Cotton effect, and the other (cystine) is probably the result of an unusually high levorotation (vide infra). Recently, Katzin and Gulyas (1964) solved equation (4)with the aid of a computer program and found that k 1 and k z of the ten amino acids studied have opposite signs, and further, the magnitudes of k l and k z , and XI and Xz are very close. Most of their X1 values varied around 200205 mp, which is slightly lower than our 210 mp; this in turn results in the difference in Xz. Their k l and kt values, however, are about an order of magnitude higher than ours. This is not surprising, since according to equation (4),[MI for the amino acids is a comparatively smaW number obtained from the difference in two large numbers. That the same compound can be fitted with two or more sets of X1 and Xz indicates that such solution of equation (4)is by no means unique. We chose X1 = 210 mp because of its physical significancein relation to the experimentally observed Cotton effect. The experimental measurements can be read to three significant figures; therefore we accordingly list in Table I only three figures. Third, in contrast with XI and 12,the X, values of the one-term Drude equation vary widely among the amino acids studied. Furthermore, the wavelength range for which equation (2) is applicable is rather narrow, except that for cystine and asparagine which extends to below 300 mp. We can convert the twoterm Drude equation into one term through powerseries expansion, provided that the following condition is satisfied (Moffitt and Yang, 1956): ki(Xi2

-

-

Xc')

k'(X2'

-

A,')

O

(7)

+ k,)

(8a) (8b)

=

Thus we have Xc2

Xc'

= (klX12

= hi2

or

+

-k kZX22)i(kl

-

k9(X2'

+ k,)

X12)/(ki

+

X,2 = A22 k,(XI' - X J ' ) / ( k , + K?) (8c) Letting X1 > X2, which is true in our case except for tyrosine, tryptophan, and cystine (Table I), we can consider two general cases: (1)k I and k? have the same sign. Obviously, X, will always be an intermediate between X1 and Xz; that is, XI > A, > A B , as can easily be deduced from equations (8b) and (8c); (2) k1 and kz have the opposite sign (irrespective of which is positive or negative). Again from equations (8b) and (812) we can show that

A,

> x1 >

Xe

< x2
XZ,

Ikll > lkzl

and hl,

if

x1 > X2,

I k l J < jk2j

(9)

ROTATORY DISPERSION OF AMINO ACIDS

1523

(see also Levene and Rothen, 1936). Furthermore, X, can differ significantly from X1 and XZ, even though the latter two may not be far apart, as long as ( k l k,) in the denominator of equation (9) is small and approaches zero. Thus in Table I we see that the A, of proline is lower than X I and hz, whereas the reverse is true for the other amino acids, in full agreement with the rules stated in equation (9). Unlike the other amino acids studied, tyrosine and tryptophan have an additional Cotton effect near 275 mp due to the aromatic chromophore. Thus, we need three Drude terms: [MI = ki/(Xz - h2) k?/(X2 - Xz2)

+

+

+ k d ( X 2 - A?)

(10)

to account for the 210- and 275-mp Cotton effects and the rest of the partial rotations. From Figure 4 we know that k1 and kz must be positive because the Cotton effects are positive. Here K 3 is expected to be negative just as in the case of other amino acids (Table I). It is therefore not too surprising to find that in Table I k 1 becomes negative and kz positive when we force the experimental data of both tyrosine and tryptophan into a two-term Drude equation (4). We can preset XI = 275 mp in equation (4))instead of 210 mp. By so doing we found that for tyrosine X1 = 275 mp, Xz = 50 ml, k1 = 5.8 X lo6, and k z = -14.6 x 106; for tryptophan X1 = 275 mp, XS = 130 mp, kl = 14.7 X lo6, and kz = 15.1 X lo6. Obviously, it has little physical meaning to force the data into a two-term equation when they call for a three-term equation. Cystine is another unusual compound; its molar rotations due to the 210 mp Cotton effect are outweighed by the rest of the partial rotations so that the total rotations appear to obey the one-term Drude equation down to 290 mp. Its A, of 207 mp is so close to 210 mp that fitting the data with equation (4) gives "abnormal" results. Note also that the inflection point of the Cotton effect in this case is closer to 215 mp than to 210 m p . For asparagine, XI and Xz happen to coincide; the one-term Drude equation is thus applicable down to 260 mp. The fact that phenylalanine has unusually large magnitude of k l and k 2 is probably related to the closeness of X I and Xz, in a way similar to the findings of Katzin and Gulyas (1964). Since this study is confined to free amino acids a t low pH only, there is of course no direct relation between rotations reported here and the residue rotations in a polypeptide chain. However, the conversion of a free amino acid to a peptide unit is equivalent to a double substitution (Schellman, 1960), and therefore the shifts in the sign and magnitude of such conversion might be expected to follow the Kuhn-Freudenberg rule. I n proteins the partial rotations of different amino acid residues usually can be assumed to approach a mean value. This rule will hold as long as the protein molecules do not contain unusually high percentages of tyrosine, tryptophan, and cystine, as our result suggests. ACKNOWLEDGMENT We thank Professor S. Beychok and Dr. J. Brahms for providing the circular dichroism data, Dr. W. B. Gratzer for helpful discussions, and Drs. L. I. Katzin and E. Gulyas for sending us their paper prior to publication. REFERENCES Billardon, M . (1960),Compt. Rend. 251, 535, 1759,6141. Brand, E., Washburn, E., Erlanger, B. F., Ellengoben, E., Daniel, J., Lippmann, F., and Scheu, M. (1954),J. A m . Chem. SOC.76, 5037.

1524

Biochemistry

J. A. RUPLEY

Calvin, M. (1954), U.S. Atomic Energy Comm., UCRL2438, p. 3 (quoted by Schellman, 1960). Chandrasekhar, S. (1952), ,. Proc. Indian Acad. Sci. Sect. A. 35,103. Katzin, L. I., and Gulyas, E. (1964), J. Am. Chem. Soc. 86, 1655. Levene, P. A., and Rothen, A. (1936), J. Org. Chem. I, 76. Moffitt, W., and Yang, J. T. (1956), Proc. Nutl. Acad. Sci. U.S. 42, 596. Moscowitz, A. (1960), in Optical Rotatory Dispersion, Djerassi, C., ed., New York, McGraw-Hill, chap. 12. Otey, M. C., Greenstein, J. P., Winitz, M., and Birnbaum, S. M. (1955), J. Am. Chem.Soc. 77, 3112. Patterson, J. W., and Brode, W. R. (1943), Arch. Biochem. 2, 247.

Samejima, T., and Yang, J. T. (1964), Biochemistry 3, 613. Sasisekharan, V. (1962), 2. Physik. Chem. 29, 219. Schellman, J. A. (1960), in Optical Rotatory Dispersion, Djerassi, C., ed., New York, McGraw-Hill, p. 210. Strem, J., Krishna-Prasad, Y. S. R., and Schellman, J. A. (1961), Tetrahedron 13, 176. Turner, J. E., Bottle, R. T., and Haurowitz, F. (1958), J.A m . Chem. Soc. 80,4117. Umes, P., and Doty, P. (1961), Aduun. Protein Chem. 16, 401. Wiirz, H., and Haurowitz, F. (1961), J. Am. Chem. Soc. 83, 280.

Comparison of Protein Structure in the Crystal and in Solution. I. The Tyrosyl Ionization of Crystalline Methemoglobin* J. A. RUPLEY From the Department of Chemistry, University of Arizona, Tucson Received June 15, 1964 The tyrosyl ionization of horse methemoglobin was measured spectrophotometrically in solution and in crystal suspensions. No difference was observed for solution and crystal maintained a t the same ionic strength, showing that upon crystallization there is no change in structure about the tyrosines. However, the tyrosyl pK of soluble methemoglobin increased with increasing ionic strength, suggesting there may be conformational changes in passing from physiological conditions to those under which crystallization occurs. No electrostatic interaction was observed for the crystalline protein, presumably a result of the high ionic strength of the buffer. Hydrogen-ion equilibria in the crystal were established rapidly and reversibly, and were normal, which indicated that no fundamental impediment exists for comparison of crystal and solution structures through comparisons of their chemical reactivities. The recent determination of the three-dimensional structure of crystalline myoglobin and hemoglobin (Kendrew, 1962; Cullis et al., 1961-62) has greatly deepened our general understanding of proteins. However it is the structure of a given protein in solution which is necessary for the detailed understanding of its properties, and to this end some assessment must be made of changes in conformation attending crystallization. Richards (1963) has thoroughly reviewed the relevant data, which, although largely indirect, strongly suggest that extensive changes are unlikely. Nevertheless, small rearrangements in structure cannot be excluded, particularly in view of the flexibility of a protein molecule while part of the crystal (Richards, 1963), and in view of the small free-energy changes associated with denaturation and structural fluctuations (Linderstrom-Lang and Schellman, 1959). One approach to defining the extent of these rearrangements is through quantitative determination of the chemical reactivities of the same groups in the crystal and in solution. This paper reports a study of the ionization of the two titratable tyrosines of crystalline and soluble horse methemoglobin, a comparison which does not detect any conformational change in crystallization. Since the demonstration of structural changes by studies of this sort is analogous to the determination of protein homogeneity, the more experiments there are which quantitatively compare the special reactivities of specific groups, the more closely the limits of putative conformational changes can be

* This investigation was supported by a research grant (GM-9410) from the Division of General Medical Sciences, U. S. Public Health Service.

set. The data below then represent only one of several necessary approaches, and lead only to conclusions concerning the environment of the tyrosines. EXPERIMENTAL Materials.-Red blood cells were harvested from fresh' citrated horse blood, washed five or more times with 0.9% saline, and laked; the cell debris was removed by centrifugation. The resulting solution of oxyhemoglobin was converted to one of methemoglobin by the addition of 1.5 equivalents of potassium ferricyanide, and was then exhaustively dialyzed against cold deionized water. After dialysis the stock solution contained 50-100 mg/ml hemoglobin and was of p H 6.6-6.7, the pH of the isoionic protein ( C o b et al., 1937). It was stored frozen. The extinction coefficient, used for routine concentration determination, was determined by drying at 103" to be Eb&,bmp= 5.12. Inorganic chemicals were reagent grade, and deionized water was used throughout. Preparation of Crystalline Methemoglobin.-In early experiments crystals were grown by dialysis against 2 M (NHa)&30a,conditions like those used by Perutz and co-workers in preparing material for crystallographic analysis (Cullis et al., 1961-62). The resulting suspensions were exhaustively dialyzed against 2 M Na2S04 to remove ammonium ions which would interfere with the titrations. No disruption of the crystal or change in habit was observed. In later experiments crystals were grown by adding methemoglobin stock solution to Obtained at the slaughterhouse of the Tucson Dog Food Co., through the courtesy of Mr. Dalrymple.